EP1097238A1 - Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres - Google Patents

Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres

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Publication number
EP1097238A1
EP1097238A1 EP99939986A EP99939986A EP1097238A1 EP 1097238 A1 EP1097238 A1 EP 1097238A1 EP 99939986 A EP99939986 A EP 99939986A EP 99939986 A EP99939986 A EP 99939986A EP 1097238 A1 EP1097238 A1 EP 1097238A1
Authority
EP
European Patent Office
Prior art keywords
clavulanic acid
application
fermentation
amino acids
free amino
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99939986A
Other languages
German (de)
English (en)
Inventor
Wilhelmus Theodorus Antonius Maria De Laat
Preben Krabben
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Koninklijke DSM NV
Original Assignee
DSM NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DSM NV filed Critical DSM NV
Priority to EP99939986A priority Critical patent/EP1097238A1/fr
Publication of EP1097238A1 publication Critical patent/EP1097238A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the present invention relates to the field of the fe'mentative production of secondary metabolites from a Streptomyces strain on a 10 suitable medium comprising carbon and nitrogen sources.
  • Clavulanic acid can be produced in substantial amounts and production conditions are being optimized continuously in order to
  • clavulanic acid has been optimized with respect to continuous fermentation (GB 1 508977), feeding of carbon to a batch process (EP- 1 82522), maintaining ammonia at low concentrations (WO 96/1 8743 and Romero J., Liras P. and Martin J.F., Appl. Microbiol. Biotechnol. ( 1 984),
  • the maximum specific growth rate is ⁇ 0.05 h "1 , Brana ( 1 986) v.s., and Aharonowitz Y. and Demain A.L., Can J. Microbiol. (1 979), Vol. 25, 61 -67, while the maximum specific growth rate is > 0.05 h "1 in media containing at least one amino acid like asparagine, aspartate, giutamate, glutamine, alanine, histidine, proline, threonine or arginine. Therefore, one would prefer the use of a medium containing one or more amino acids. In such media, the production of biomass takes less time compared to inorganic media containing only ammonia which is of course an advantage over a slower production process.
  • Asparagine is described in the non-prepublished International application WO 98/371 79 as fermentation ingredient for the production of clavulanic acid and also for cephalosporin, e.g. cephamycin C production from Streptomyces clavuligerus (Aharonowitz Y. and Demain A.L., v.s.) .
  • cephalosporin e.g. cephamycin C production from Streptomyces clavuligerus (Aharonowitz Y. and Demain A.L., v.s.) .
  • giutamate was shown to repress clavulanic acid formation in a batch process (Romero (1 984), v.s.), one would not be directed to use giutamate or glutamine.
  • hydrolysed proteins preferably rich in giutamate and proline in the fermentation broth of a secondary metabolite producing Streptomyces and the particular advantage of gluten hydrolysate and casein hydrolysate in this respect.
  • a source of amino acids in particular giutamate as a feeding nutrient in a fed batch process for the production of these compounds.
  • Figure 1 titre of clavulanic acid during a batch fermentation at low levels of free giutamate.
  • Figure 2 titre of clavulanic acid during a fed batch fermentation at low levels of free giutamate.
  • the present invention provides a method for the production of secondary metabolites by the fermentation of such a secondary metabolite producing Streptomyces on a suitable medium by keeping the concentration of free amino acids lower than 5 g/l fermentation broth, preferably lower than 2.5 g/l and more preferably lower than 0.5 g/l, provided that a concentration of 4 g/l free asparagine supplied Jo a fermentation broth of Streptomyces clavuligerus has been excluded.
  • This process is especially favourable for the production of -lactams, polyketides and macrolides, preferably clavulanic acid, pimaricine, erythromycine, nystatine or amphotericine.
  • one or more of the amino acids preferably giutamate or proline. is applied in a fed-batch or continuous mode.
  • amino acids preferably giutamate or proline.
  • protein hydrolysate more preferably glutenhydrolysate or caseinhydrolysate and most preferably wheat glutenhydrolysate is applied in a batch, fed-batch, semi-continuous or continuous mode.
  • the present invention describes the use of media poor in free amino acids, by the application of said amino acids in a fed batch or continuous mode or by the application of protein hydrolysate in any mode.
  • a protein is defined as a polymer of amino acids with a size expressed by a Molecular Weight > 20,000 Dalton which has not been processed by any means to degrade the protein to smaller fragments.
  • a protein hydrolysate is defined to be a polymer of amino acids with an average size between 300 Dalton and 20,000 Dalton and a free amino acid content of less than 30% of the total amino acids.
  • These protein hydrolysates can be produced either by enzymatic or by chemical hydrolysis of the corresponding proteins. Alternatively the advantage of using hydrolyzed proteins is achieved by using strains improved for protease activity in a process using non- hydrolyzed proteins as raw materials.
  • giutamate stands in the present application for glutam like compounds as giutamate and glutamine.
  • a protein extract is defined to be a protein hydrolysate with a molecular weight lower than 300 Daitorx and wherein > 30% of the amino acids are present as free amino acids.
  • a protein or the hydrolysate thereof is defined as rich in giutamate it is meant that > 1 5% of the amino acid content consists of giutamate and glutamine. Proteins described as rich in giutamate are casein (21 %) and wheat gluten (35%).
  • clavulanic acid production was especially high when a protein hydrolysate was included in the medium, especially when it was derived from wheat gluten or casein.
  • protein hydrolysates should preferably contain less than 30% free amino acids, even more preferably less than 5 % and most preferably less than 1 % free amino acids.
  • a protein hydrolysate preferably rich in giutamate can be used as well supplied in all forms of feeding, viz. batch, fed batch, continuous or semi-continuous.
  • semi-continuous feeding is meant the continuous addition of nutrients to the fermentation broth while intermittently a small volume of the broth is removed.
  • the fermentation medium may either be a defined medium comprising (NH 4 ) 2 SO 4 , free amino acid, KH 2 PO 4 , MgSO 4 .7H 2 O, CaCI 2 .2H 2 O, 3-(N-morpholino), propanesulfonic acid, glycerol, sodium succinate and a solution of trace elements, with a low concentration of amino acid, or with a protein hydrolysate which results in a complex medium.
  • a complex medium as for instance flours from nuts, vegetables, seeds, cereals, grasses such as those useful in fermentation industry, soybean flour, lineseed flour, peanut flour, potato flour, sunflower, pea- or beanflour, cotton seed flour, wheat gluten, whole wheat, rice meal to which protein hydrolysate is added, wherein the concentration of free amino acid is low, is part of the invention.
  • the medium may also contain mixtures of mentioned flours with mixtuRes of protein hydrolysates of various sources and of various peptide sizes as desired to achieve the optimal result.
  • microbial strains which are capable of being fermented in a chemically defined medium and/or improved by subjecting a parent strain of interest to a classical mutagenic treatment using physical means, such as UV irradiation, or a suitable chemical mutagen, such as N-methyl-N'-nitro-N-nitrosoguanidine or ethylmethane sulfonate may be used for the process of the present invention.
  • a parent strain of interest to recombinant DNA technology, whereby the parent strain is transformed with a one or more functional genes of interest.
  • Any assimilable carbon source may be added to the above said mixture, like sugars such as glucose, fructose, sucrose, maltose, lactose, or polysaccarides like starch, maltodextrines and inuline or other fructose polymers, triglycerides such as soybean oil, sunflour oil, olive oil, tri-oleate etc., (poly-) alcohols such as ethanol, propanol, glycerol, mannitol, or organic acids or a salt thereof such as acetate, propionate, succinate, adipate, malonate, citrate, lactate, gluconate etc.
  • sugars such as glucose, fructose, sucrose, maltose, lactose, or polysaccarides like starch, maltodextrines and inuline or other fructose polymers
  • triglycerides such as soybean oil, sunflour oil, olive oil, tri-oleate etc.
  • An inorganic nitrogen source may be added to the medium such as ammonia and/or nitrate or any of its salts. Ureum may be used as well. Furthermore also vitamins, and various sorts of inorganic anions such as sulphates, phosphates, chlorides, borates, molybdate, iodate or their salts and the cations potassium, sodium, zinc, manganese, magnesium, iron, copper, cobalt, nickel etc. may be added to the medium.
  • a fermentation is started by inoculating from a preculture or inoculum fermentation at a volume of about 1 to 50% of the main fermentation medium, particularly from 5 to 20%.
  • the process may last from about 24 to 400 hours and especially from 48 to 1 68 hours.
  • the temperature will be kept between 20 and 40 °C, preferably between 25 and 35 ° C, and even more preferably between 26 and 30 ° C.
  • the pH can be maintained at pH 6 to 8 by means of titration with an alkaline substance such as ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or an organic base like lysine, arginine and histidine and an acid substance, such as the anorganic acids like sulphuric acid and hydrochloric acid.
  • an organic acid may be used such as giutamate, citrate, gluconate or acetate.
  • the dissolved oxygen concentration may be controlled in the optimal range for the process by varying the oxygen concentration in the inlet gas, application of overpressure, modification of stirrerspeed and airflow.
  • the range may vary between 0 and 1 00% of air saturation.
  • the process may be carried out by controlling various non-growth limiting nutrients in their optimal concentrations.
  • these growth-non-limiting nutrients may contain any relevant carbon, nitrogen, phosphor, sulphur source or oxygen.
  • Carbon dioxide should be kept at non-toxic concentrations by for instance increasing the airflow through the fermentor so that the carbon dioxide concentration in the outlet-gas is less than 5%, preferably less than 2.5%.
  • the fermentation can be carried out in a batch, fed batch, or (semi-) continuous fermentation process mode.
  • the recovery of the impure clavulanic acid solution as formed by the fermentative process of the present invention as well as the subsequent conversion thereof into a pharmaceutically acceptable salt by methods known in the art do form an aspect of the present invention.
  • One of the most advantageous procedures is the conversion of the impure clavulanic acid into an amino salt thereof by adding the corresponding amino salt forming compound as for instance N,N,N',N'- tetramethylethylenediamine, 1 ,3-bis(di-methylamino)-2-propanol, t- butylamine, t-octylamine, benzhydrylamine and bis (2-(dimethyl- amino)ethyl)ether and reacting said amine clavulanate with a non-toxic pharmaceutially acceptable salt as for instance potassium ethylhexan emerge to form the corresponding purified salt, for instance potassium clavulanate.
  • the corresponding amino salt forming compound as for instance N,N,N',N'-
  • Streptomyces clavuligerus ATCC27064 was improved for clavulanic acid production by means of several rounds of classic mutation (UV, nitroso guanidine (NTG)) and selection in shake flask cultures whereby clavulanic acid production was tested by imidazole methods as known in the art.
  • the strain was conserved as vegetative mycelium grown for 48 hours in Tryptone Soytone Broth-medium (TSB-medium) at 28 ° C in a shaker incubator shaken at 280 rpm and stored frozen at -80 °C.
  • 1 ml of the frozen mycelium was inoculated to 1 00 ml of a sterilized (30 minutes, 1 21 °C) preculture medium containing 5-20 g/l maltose.1 aq, 1 5-30 g/l bacto tryptone, 1 5-30 g/l bacto peptone, 1 -1 0 g/l bacto soytone, mono potassium phosphate 1 -5 g/l and 0.2 g/l synthetic antifoam.
  • a sterile production medium containing 2.5 g/l from a complex nitrogen source such as a protein, a protein hydrolysate and/or or protein extract.
  • the production medium further contains 50- 1 00 g/l glycerol, 5-20 g/l soybean flour, 0.5-2 g/l mono potassium phosphate, a suitable trace element cocktail and 0.2 - 2 g/l synthetic antifoam.
  • Table 1 Clavulanic acid production with a mutant strain from S. clavuligerus ATCC 27064 using different complex nitrogen sources additional to soybean flour.
  • Batch fermentation of clavulanic acid Streptomyces clavuligerus ATCC 27064 was precultivated for 26 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM on a medium containing 5 to 30 g/l glycerol, 5 to 30 g/l soy peptone, 2 to 6 g/l sodium chloride, and 0.5 to 3 g/l calcium carbonate.
  • the preculture was inoculated at a volume of 1 0 % into 1 I of chemically defined medium containing 1 0-30 g/l glycerol, 0.5 to 3 g/l KH 2 PO 4 , 1 to 3 g/l (NH 4 ) 2 SO 4 , 1 5-25 g/l monosodium giutamate, 0.05 to 0.2 g/l FeSO 4 -7 H 2 O, 0.1 to 1 g/l MgSO 4 -7 H 2 O, 1 0-20 g/l 2-(N- morpholine) propane sulfonic acid (MOPS), 0.2-1 g/l basildon antifoam, and a suitable trace element solution.
  • the pH was adjusted to 6.8 with 4N NaOH.
  • the second preculture was cultivated for 20 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM.
  • the second- preculture was inoculated at a volume of 3.3 % into 29
  • the fermentation was carried out at 30°C and the pH was controlled at 6.95 to 7.05 by titration with 4N NaOH and 4N H 2 SO 4 .
  • the dissolved oxygen tension was maintained above 50 % of air saturation or regulated at 50 % of air saturation by the stirrer speed.
  • Streptomyces clavuligerus ATCC 27064 was precultivated for 26 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM on a medium containing 5 to 30 g/l glycerol, 5 to 30 g/l soy peptone, 2 to 6 g/l sodium chloride, and 0.5 to 3 g/l calcium carbonate.
  • the preculture was inoculated at a volume of 1 0 % into 1 I of chemically defined medium containing 1 0-30 g/l glycerol, 0.5 to 3 g/l KH 2 PO 4 , 1 to 3 g/l (NH 4 ) 2 SO 4 , 1 5-25 g/l monosodium giutamate, 0.05 to 0.2 g/l FeSO 4 -7 H 2 O, 0.1 to 1 g/l MgSO 4 -7 H 2 O, 1 0-20 g/l 2-(N- morpholine) propane sulfonic acid (MOPS), 0.2-1 g/l basildon antifoam, and a suitable trace element solution.
  • the pH was adjusted to 6.8 with 4N NaOH.
  • the second preculture was cultivated for 20 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM.
  • the second preculture was inoculated at a volume of 4 % into 24 I of a chemically defined medium containing 5 to 1 5 g/l glycerol, 0.5-2 g/l
  • the fermentation was carried out at 30°C and the pH was controlled at 6.95 to 7.05 by titration with 4N NaOH and 4N H 2 SO 4 .
  • the dissolved oxygen tension was maintained above 50 % of air saturation or regulated at 50 % of air saturation by the stirrer speed.
  • a carbon feed containing 300 to 500 g glycerol/kg feed was added at a rate of 30-60 g/h and a nitrogen-phosphate-feed containing 50 to 1 00 g Na-glutamate/kg feed, 5 to 10 g (NH 4 ) 2 SO 4 / kg feed, and 5 to 1 0 g K 2 HPO 4 /kg feed was added at a rate of 50 to 1 50 g/h.
  • Clavulanic acid and giutamate concentrations in the medium are demonstrated in figures 1 and 2 respectively for the batch process and the fed batch process. From figure 1 it can seen that clavulanic acid production starts when the giutamate concentration is dropping below 5 and even more preferably below 2.5 g/l and ends at 250-300 mg clavulanic acid /liter.
  • Figure 2 shows that when giutamate is fed to the fermentor keeping the concentration very low ( ⁇ 1 g/L) after 30 hour ⁇ .
  • clavulanic acid titers can increase to 500 mg/L in this experiment, giving a doubling compared to the batch experiment.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
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  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé amélioré visant la production de métabolites secondaires par la fermentation d'une souche de Streptomyces. Si la concentration d'acides aminés libres reste faible, on obtient un rendement étonnamment élevé.
EP99939986A 1998-07-03 1999-07-01 Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres Withdrawn EP1097238A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99939986A EP1097238A1 (fr) 1998-07-03 1999-07-01 Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP98202264 1998-07-03
EP98202264 1998-07-03
EP99939986A EP1097238A1 (fr) 1998-07-03 1999-07-01 Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres
PCT/EP1999/004702 WO2000001840A1 (fr) 1998-07-03 1999-07-01 Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres

Publications (1)

Publication Number Publication Date
EP1097238A1 true EP1097238A1 (fr) 2001-05-09

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Application Number Title Priority Date Filing Date
EP99939986A Withdrawn EP1097238A1 (fr) 1998-07-03 1999-07-01 Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres

Country Status (7)

Country Link
EP (1) EP1097238A1 (fr)
CN (1) CN1308682A (fr)
AU (1) AU5409799A (fr)
CA (1) CA2336363A1 (fr)
MX (1) MXPA00012930A (fr)
TR (1) TR200003828T2 (fr)
WO (1) WO2000001840A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1122305A1 (fr) * 2000-01-31 2001-08-08 Dsm N.V. Streptomyces clavuligerus transformé par la glutamate déshydrogénase présentant une capacité améliorée d'assimiler de l'ammoniac
WO2009138994A1 (fr) * 2008-05-14 2009-11-19 Biocon Limited Procédé de fermentation pour la fabrication de métabolites secondaires
KR101729251B1 (ko) * 2015-04-28 2017-04-21 주식회사 대웅 보툴리눔 독소의 제조를 위한 배지 조성물
KR101723167B1 (ko) * 2015-04-28 2017-04-05 주식회사 대웅 보툴리눔 독소의 제조를 위한 배지 조성물
CN105817190B (zh) * 2016-03-15 2019-01-08 浙江凯色丽科技发展有限公司 水解自动化控制系统
CN111944734B (zh) * 2019-05-17 2023-01-10 中国科学院微生物研究所 提高链霉菌聚酮化合物产量的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE41109B1 (en) * 1974-04-20 1979-10-24 Beecham Group Ltd Novel -lactam antibiotic from streptomyces clavuligerus
AT353409B (de) * 1975-02-07 1979-11-12 Glaxo Lab Ltd Verfahren zum entfernen von verunreinigungen aus clavulansaeure oder deren salzen
EP0026044B1 (fr) * 1979-08-24 1983-06-08 Beecham Group Plc Sel amine de l'acide clavulanique, sa préparation et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0001840A1 *

Also Published As

Publication number Publication date
AU5409799A (en) 2000-01-24
CN1308682A (zh) 2001-08-15
CA2336363A1 (fr) 2000-01-13
TR200003828T2 (tr) 2001-06-21
MXPA00012930A (es) 2002-05-08
WO2000001840A1 (fr) 2000-01-13

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